estrogenic effect of orthopedic - egyptianjournal.xyzegyptianjournal.xyz/2_9.pdf · tion of ddt...

The Egyptian Journal of Hospital Medicine Vol., 2 : 108 – 120

March, 2001 I.S.S.N: 12084

Estrogenic Effect of Orthopedic

DDT on the Female Genital

Organs of Mice

Raifa F. El Garieb National Centre for Radiation Research and Technology

Abstract :

Insecticide DDT (an organochlorine compound) has been used excessively and

widely all over the world. It was shown that orthopedic DDT (o-p'-DDT) a major

constituent of technical grade preparation of the pesticide- DDT exhibited estrogenic

activity in several animals species (Welch et al., 1969 and Bitman et al., 1968).

The aim of this work is to study the estrogenic effects of o-p'-DDT on the female

reproductive organs (ovary, uterus and vagina) of mice in its neonatal period.

In this study fifty of newly-born female mice were used and divided into five

groups; three groups of them were received ten intraperitoneal injections of o-p'- DDT

in doses of 0.25, 0.5 and 1mg/injection. The study included two control groups, the first

one was injected with 10 ugm estrogen for 10 injections and considered as positive

control, while the second group injected with sesame oil only. The animals were

scarified 2 days after the last injection. Paraffin and frozen sections were prepared for

both histological and histochemical studies. Different staining techniques were used

including Hx., E., modified gomori stain for alkaline phosphatase enzyme, methyl green

pyronin for RNA and PAS stain for glycogen content.

The results showed a distinct increase in number and size of ovarian growing

follicles in those animals treated with o-p'-DDT, also, increased activity of the alkaline

phosphatase enzyme, as well as increased content of RNA and glycogen was noticed.

The effect of o-p'-DDT on the uterine structure were in the form of hypertrophy of the

endometrial epithelium, also increase in number of uterine glands. Marked increase in

the activity of alkaline phosphatase enzyme and also in RNA content as well as

glycogen deposition.

It was noticed also that high dose of o-p'-DDT leads to accelerated development

of the vaginal epithelium with evidence of keratinization. The histochemical changes in

the vagina were similar to those obtained in the uterus.

It can be finally concluded that the obtained results after use of o-p'-DDT were

similar to those obtained by estrogen, and this may interfere with implantation of ova

and pregnancy.

Refree : Prof . Moustafa Ismail.H.

Raifa F. EL Garieb

109

Introduction :

The toxicity of various pesticides is of

general importance because of their

wide use in agriculture. While these

pesticides destroy insects, they may

have a direct action on the reprod -

uction of birds and mammals confined

to the treated fields. Diminished reprod

-uction of various species following

consumption of diets containing resid -

ues of DDT (organochlorine compound)

have been suggested by several reports

(Bitman et al., 1968; Welch et al., 1969;

and Kupfer & Bulger, 1976).

It has found that DDT and its

closely related compounds exhibited

estrogenic effects (Heinrichs et al.,

1971). Technical commercial prepara -

tion of DDT contained o-p'-DDT and

p,p'-DDT isomers, constituting about

20% to 80% of the pesticide prepa -

ration, respectively. Reports indicate

that o-p'-DDT and p-p'-DDT mimic

various hormonal actions of oestrogen

in various species of birds and mam -

mals (Bitman et at, 1968).

In birds and mammals estrogen

plays a critical role in female reprodu -

ction. In mammals, structural changes

in vagina, uterus and 'duct are affected

by estrogens and proper ovarian stru -

cture and ovarian structure and funct -

ion becomes critical for reproduction

(Turner and Bagnara, 1976).

Treatment of immature chickens,

rat and Japanese quail with o-p'-DDT

produced an increase in weight, water

content, glycogen and RNA in uteri and

oviducts (Levin el aL, 1968; Conney

and Btirns, 1972).

The o-p'-DDT isomer also

advanced puberty, induced persistent

estrus and caused ovaries to develop

follicular cysts and reduced the number

of corpora lutea in rats (Heinrichs et aL,

1971; Wren et aL, 1971).

Injection of o-p'-DDT into neon

-atal rats produced vascularization and

hypertrophy of the cells in the endomet

-rium, metaplastic changes which interf

-ere with implantation of the fertilized

ova, these findings and the decrease of

gonadotropins suggest that neonatal exp

-osure to estrogenic pesticides such as

o-p'-DDT may permanently affect the

sensitivity of hypothalamic-hypophysial

axis and the uterus to endogeneous

estrogen (Gellert et aL, 1972; 1974).

Estrogen plays an important role

in increasing the activity of alkaline

phosphatase activity in female genital

organs (Filipe and Dowson, 1968) also

it was found that estrogen produced an

increase in the endometrial mucopoly -

saccharide concentration (Huber, 1965).

The functional relationship betw -

een DNA and RNA mediated protein

synthesis was under the control of

estrogen (Edwards 1967).

Laguens (1964) suggested that

estrogen act upon the nuclear synthesis

and secondary release of nuclear RNA

into the cytoplasm.

It was reported that human bre -

ast milk is contaminated with DDT and

its analogs (Wilson et aL, 1973).

Another report indicates that o-

p'-DDT and p,p-DDT are not only

found in mother milk but also in baby's

formula milk and consequently the

babies are exposed to the same toxic

hazards as breast fed babies (Cockson

and Morgan, 19 76).

The passage of estrogenic pestic

-ides through mother's milk to the

developing offspring will have a critical

influence on the development of female

as well as male reproductive organs.

108


110

Current studies indicate that there is a

critical period in the development

during which the undifferentiated

hypothalams is sensitive to gonadal

hormones in circulation. In species

such as rat or mouse, the sensitivity

period is up to 8 days afterbirth (Turner

and Bagnara, 1976). It was felt advis -

able that the stud of the mechanism of

action of DDT and its effects on the

reproductive systems of different spec -

ies of animals in neonatal period is

highly indicated.

This study was planned to

evaluate the morphological and histoc -

hemical changes after the use of o-p'-

DDT on the female reproductive organs

namely (ovary, uterus and vagina) early

in the post-natal life which represent the

critical period in the development of

such organs.

Material and Methods:

In this study fifty Swiss-Webster

female mice were used and divided into

5 groups; three groups were injected

intraperitoneally with o-p'-DDT in a

doses of 0.25 mg, 0.5 mg and I mg

/injection dissolved in 0. I ml sesame oil

for ten days, the fourth group received

10 injections of 10 ug/injection of estr -

ogen, while the last group received sesa

-me oil only and considered as control.

Theinjection started on the first day

after birth. The animals were termin -

ated 2 days after the last injection.

The entire reproductive organs

from each female mice were removed,

paraffin and frozen sections were prep -

ared and cut for both morphological and

histochemical studies.

Different staining techniques

were used; heamatoxylin and eosin

(Culling.1974) for the morphological

changes, modified Gomorl stain for

localization of the alkaline phosphatase

enzyme activity (Pearse 1968). Methyl

green pyronin stain for staining the

RNA (Drury and Walington,1980) and

finally PAS stain for study the glycogen

content (Drury and Walington, 1980).

Results :

(I) The morphological results : a- Ovary : It was noticed that ovaries from a

12-day-old mouse which injected with

sesame oil only showed a large number

of primary oocytes and few number of

small growing follicles. After injection

with o-p'-DDT in doses of 0.25 mg and

0.5 mg/injection for ten injections the

ovaries were characterized by mild

increase in the number of growing

follicles. However, a distinct increase

in both the number and the size of

growing follicles was evident in ovaries

after the injection with 1mg/injection of

o-p'-DDT for ten days specially when

compared with those of the control

ovaries (Fig. 1,2).

b- Uterus : In this study, it was noticed that

the endometrial changes was variable.

After injection with o-p'-DDT in doses

of 0.25 mg or 0.5 mg/injection ednom

etrial epithelial hypertrophy was noticed

together with increase number of

glands. After the injection of 1 mg/inje-

ction of o-p'-DDT for ten days the

endometrial epithelium was of tall

columnar type with numerous glands

(Fig. 3,4).

c- Vagina : In this study, the vaginal

epithelium of a 12-day-old mouse injec

-ted with sesame oil only was formed of

two or three cubodial cells resting on

loose stromal C.T. After the injection of

o-p'-DDT in doses of 0.25 mg and 0.5

mg/injection for 10 days, thickening of

vaginal epithelium was a prominent

feature, while daily injection of 1 mg o-

p'-DDT for ten days induced a more

Raifa F. EL Garieb

111

rapid development of vaginal epithe

lium which exhibited a distinct basal

cell layer, stratum spinosum and

stratum granulosum with dark-staining

keratohyaline granules and extensive

keratinization. The vaginal canal contai

-ns desquamated keratinized cells with

leucocytes (Fig. 5,6).

(II) The histochemical results :

a- Changes in the alkaline phosphatase

enzyme :

It was noticed that the injection

of o-p'-DDT in its different doses

greatly affect the activity of alkaline

phosphatase enzyme also, such enzy -

matic changes were dose related.

A definite increase in alkaline

phosphatase activity was noticed in the

ovarian follicles, epithelial lining of

both uterus and vagina together with the

endometrial glands (Fig. 7-12).

b- Changes in the ribonucleic acid

(RNA) content :

It was noticed that the injection of

o-p'-DDT does not affect the DNA

content in the different organ studied.

While the RNA content was greatly

affected after the injection of different

doses of such insecticide. The amount

of RNA was noticed to be increased in

the ovarian follicles, lining epithelium

of both uterus and vagina together with

cells of the endometrial glands. Such

changes were dose related to the

injected insecticide (Fig. 13-18).

c- Changes in the glycogen content :

Increase in the glycogen contents

in the ovarian follicles, epithelial lining

of both uterus and vagina, as well as

uterine glands was observed in animals

injected with different doses of o-p'-

DDT specially when compared with

those of the control group (Fig. 19-25).

It was noticed that morphological

and histochemical change observed in

reproductive organs of the female mice

after receiving estrogen daily for ten

days were similar to that showed in

animals injected with high dose of o-p'-

DDT.

Legand of Figures

Collective photomicrograph (A) (1-6) : Fig. (1):Control mice ovary showing

few number of growing folli -

cles (Hx. E.X 100).

Fig. (2):Mice ovary, treated with 1

mg/injection of o-p'-DDT show

-ing increased number and size

of growing follicles (Hx.E.X.

100).

Fig. (3):Control mice uterus showing

lining epithelium and few num

-ber of uterine glands (Hx.E.X

100).

Fig. (4):Mice uterus, treated with 1

mg/injection of o-p'-DDT sho -

wing tall columnar epithelium

and increased number of uteri -

ne glands (Hx.E.X 100).

Fig. (5):Control mice vagina showing

lining epithelium of 3 cuboidal

cells (Hx.E.X 250)

Fig. (6):Mice vagina veatod with 1

mg/injection of o-p'-DDT show

-ing increased epithelial stratif

-ication with keratinization

(Hx.E.X 100).

Collective photomicrograph (B) (7-12)

Fig. (7):Control mice ovary showing

distribution of alkaline phosph

-atase activity (Modified Gom

-ori, X 100)

.Fig. (8):Mice ovary, treated with 1

mg/injection of o-p'-DDT sho -

wing increased alkaline phosph

-atase activity in growing follic

-les (Modified Gomori, X 100).

Fig. (9):Control mice uterus showing

the distribution of alkaline pho


112

-sphatase activity (Modified

Gomori, X100).

Fig. (10):Mice uterus, treated with 1

mg/injection of o-p'-DDT

showing increased alkaline

phosphatase activity in surface

epithelium and uterine glands

(Modified Gomori, X 100)

.Fig. (11):Control mice vagina showing

the distribution of alkaline

phosphatase activity (Modified

Gomori, X250).

Fig. (12):Mice vagina treated with 1


showing increased alkaline pho

-sphatase activity in surface

epithelium (Modified Gomori

X 250).-

Collective photomicrograph (C) (13-18) : Fig. (13):Control mice ovary showing

the distribution of RNA

(Methyl green pyronin stain

X 100)

Fig. (14)Mice ovary njected with I


showing an increase in the

amount of RNA at the

growing follicles (1\4ethyl

green pyronin stain X 100).

Fig. (15) Control mice uterus showing


(Methyl green pyronin X

100).

Fig. (16) Mice uterus injected with I



amount of RNA at the surface

epithelium (Methyl green

pyronin stain X I 00).

Fig. (17) Control mice vagina showing


(Methyl green pyronin stain

X 250).

Fig. (18) Mice vagina injected with I

mg/inj'ection of o-p'-DDT


amount of RNA at the surface

epithelium (Methyl green

pyronin stain X 100).

Collective photomicrograph (D) (19-

24): Fig. (19) Control mice ovary showing

the distribution of glycogen

(P.A.S. stain X 100).

Fig. (20) Mice ovary treated with I

mg/inj'ection of o-p'-DDT

showing increase glycogen

content in the growing follicles


Fig. (21) Control mice uterus showing



Fig. (22) Mice uterus treated with I


showing increase glycogen

content at the lining epithelium


Fig. (23) Control mice vagina showing



Fig. (24) Mice vagina treated with 1


showing increase glycogen in

the lining epithelium (P.A.S.

stain X 400).

Raifa F. EL Garieb

113


114

Raifa F. EL Garieb

115


116

Raifa F. EL Garieb

117

Discussion

In the environment, DDT accum -

ulates gradually and persist for long

times in the bodies of exposed species.

Thus, the presence of small amounts of

DDT in environment may not cause an

immediate harm to animal life, but may

instead induce an estrogenic hazard in

which reproduction of animals is threa -

tened. The undeveloped reproductive

organs of mice serve as convenient

target for studying the estrogenic acti -

vity of DDT. The epithelium lining of

either the vagina or the uterus of the 12-

day-old mouse similar to that of an

ovarlectomized adult female (Iguchi et

aL, 1976, Mori, 1977) and was thin and

undeveloped.

Previous studies indicated that

there was a critical period in the develo

-pment during which the undifferen -

tiated hypothalamus is sensit ' ive to the

circulating gonadal hormones. In the

mouse, this period was not ended until

about 8 days after birth (Turner and

Bagnara, 1976).

In this work, the results showed

that, the neonatal administration of the

different doses of o-p'-DDT leads to an

increase in the number of the ovarian

growing follicles as well as accelerated

developmental changes in both the uter

-us and the vagina in a dose-dependent

manner, with the higher doses produ -

cing maturation in the shorter time.

These estrogenic effects of o-p'-DDT

were in agreement with those reported

by many investigators in different

species (Welch et al., 1969; Heinrichs et

al., 1971, Nigam et al., 1977 and

Aldridge 1990).

Eroschenko and Mousa, (1979)

stated that "kepone" as one of the

estrogenic pesticides-induced changes

in the reproductive tract of the female

mouse; dentical to those observed by

the estrogen.

Huber, (1965) stated that kepone

altered the ovarian structure and distur -

bed the female hormonal balance in the

adult mice.The histochemical results

obtained in this study showed an incre -

ase in the activity of the alkaline phosph

-atase enzyme, as well as in the content

of RNA and glycogen in the different

organs studied of female mice injected

with o-p'-DDT. These results were coin

-cided with those results obtained by

Mousa et al., (1984), after using the

kepone in the neonatal mouse.

Conney and Burns (1972),

described an increased weight, water

content, glycogen and RNA in uteri and

oviducts of rats and chickens when

injected with estrogen. Donald et al.,

(1965) reported that estrogen injection

stimulated the production of both

ribonucleo-proteins and alkaline phosph

-atase enzyme. Many reports stated that

estrogenic pesticides administration incr

-eased the RNA content in the uteri and

oviducts of rats, chicken and quail

(Bitman et al., 1968, Levin et al., 1968

and Beeman 1982).

Cooke (1970) described an

increase in the glycogen content of the

oviduct of immature chicks and

Japanese quail after administration of o-

p'-DDT.

The mechanism of action of o-p'-

DDT on the mammalian reproductive

system is presently not understood.

However, since o-p'-DDT induced an

estrogenic response, it may act by

interaction with estrogen receptors in

the cells of the mouse reproductive tract

as it has been demonstrated in quail

(Turnes and Eliel 1978). Jefferies,

(1967) postulated that DDT may affect

the function of the pituitary gland or the

hypothalamus.

It can be postulated from the for

mentioned results that o-p'-DDT has a


118

definite estrogenic effect which leads to

abnormalities in the ovaries and cells of

both uterus and vagina of mouse and

may interfere with ova or sperm

transport as well as the process of

implantation and pregnancy.

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120

التأثير االستروجينى للمبيد الحشرى

)ت.د.د) على الجهاز التناسلى ألنثى الفأر الصغير

رئيفه فتحى الغريب

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اظ باديلد من ب ألل ج بيدطك لرب اف هلرب ع دربرد ع ربش أريربرة قربر رب اظ ياربل باديربلد مصربلا * ع ربربلز عربربزو ب سربرب لو ب قلربرب ة ربربش ب ربربلمض ب ألربرب ب يدربرب ا بال كربرب أني ب بعربربلت ب ربربن اقألربربت

.د.د. ل رب دب غ ربل بيربدطك لةلدربل مربو تكرب يك حدقربش ا رب ش ت إع منرب خاليربل.د. ةربل د بسربتمخربم ب قربك ل ربرب د *

سربربربطح ش مصربربربح ل لاديربربربلد ع ربربربلز عربربربزو ب سربربرب لت ز ب قلربربرب ة ربربربش ب ربربربلمض ب ألربربرب ب يدربربرب ا مربربربو ايربربربلدة . بضحش ة ش بال ك أني بيرتسدش بخلاليل

لالسربرت أني مربك ب أللا ربش ت ميلثربل إع اربر دربري تربأثري ب قربك .د. قر د ت ب ألتل ج لى تأثري ب قربك ل ربرب د .بي عف أ ش بولت ة ل ش لى ال باللا ب تأللسلى بيمختل ش ل لع ب صغري

estrogenic effect of orthopedic - egyptianjournal.xyzegyptianjournal.xyz/2_9.pdf · tion of ddt...

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